Dnmt3a-CD is less susceptible to bulky benzo[a]pyrene diol epoxide-derived DNA lesions than prokaryotic DNA methyltransferases

Benzoa]pyrene (Ba]P) is a well-characterized environmental polycyclic aromatic hydrocarbon pollutant. In living organisms, Ba]P is metabolized to the genotoxic anti-benzoa]pyrene diol epoxide that reacts with cellular DNA to form stereoisomeric anti-Ba]PDE-N(2)-dG adducts. In this study, we explored the effects of adduct stereochemistry and position in double-stranded DNA substrates on the functional characteristics of the catalytic domain of murine de novo DNA methyltransferase Dnmt3a (Dnmt3a-CD). A number of 18-mer duplexes containing site-specifically incorporated (+)- and (-)-trans-anti-Ba]PDE-N(2)-dG lesions located 3'- and 5'-adjacent to and opposite the target cytosine residue were prepared. Dnmt3a-CD binds cooperatively to the DNA duplexes with an up to 5-fold greater affinity compared to that for the undamaged DNA duplexes. Methylation assays showed a 1.7-6.3-fold decrease in the methylation reaction rates for the damaged duplexes. Ba]PDE modifications stimulated a nonproductive binding and markedly favored substrate inhibition of Dnmt3a-CD in a manner independent of DNA methylation status. The latter effect was sensitive to the position and stereochemistry of the Ba]PDE-N(2)-dG adducts. The overall effect of trans-anti-Ba]PDE-N(2)-dG adducts on Dnmt3a-CD was less detrimental than in the case of the prokaryotic methyltransferases we previously investigated.